Dihydrocinnamyl phenols useful as antimicrobial agents

ABSTRACT

Substances which are subject to microbial spoilage are preserved by addition of a dihydrocinnamyl phenol, e.g., dihydro-2cinnamyl-phenol, dihydro-4-cinnamyl-phenol, dihydro-2-cinnamyl-4methylphenol, etc.

United States Patent [1 1 Jurd et al.

[451 Feb. 18, 1975 DIHYDROCINNAMYL PHENOLS USEFUL AS ANTIMICROBIAL AGENTS [75] Inventors: Leonard Jurd, Berkeley; Kenneth L.

Stevens, Walnut Creek; Alfred Douglas King, Jr., Martinez, all of Calif.

[73] Assignee: The United States of America as represented by the Secretary of Agriculture, Washington, DC.

22 Filed: Apr. 2, 1973 21 Appl. No.: 347,084

Related U.S. Application Data [62] Division of Ser. No. 257,031, May 25, 1972, Pat. No.

OTHER PUBLICATIONS Hurd et al., Jour. Am. Chem. Soc.. Vol. 59, pp. 107109.

Barnes et al., Tetrahedron, Vol. 21, pp. 2,7072,7l5.

Jurd, Experientia, Vol. 24, pp. 858-860.

Jurd, Tetrahedron, Vol. 25, pp. l,407-l,4l6.

Jurd, Tetrahedron, Letters No. 33, pp. 2.8632,866.

Thakar et al., Chem. Abst., Vol. 63 (1965), pp. 571-572.

Primary Examiner-Jerome D. Goldberg Assistant Examiner-Allen J. Robinson [57] ABSTRACT Substances which are subject to microbial spoilage are preserved by addition of a dihydrocinnamyl phenol, e.g., dihydr0-2-cinnamyl-phenol, dihydro-4-cinnamylphenol, dihydro-Z-cinnamyl-4-methylphenol, etc.

2 Claims, No Drawings DIHYDROCINNAMYL PHENOLS USEFUL AS ANTIMICROBIAL AGENTS This is a division of our copending application, Ser. No. 257,031, filed May 25, 1972, issued Nov. 27, 1973, and U.S. Pat. No. 3,775,541.

A non-exclusive, irrevocable, royalty-free license in the invention herein described, throughout the world for all purposes of the US. Government, with the power to grant sublicenses for such purposes, is hereby granted to the Government of the United States of America.

DESCRIPTION OF THE INVENTIQN This invention relates to the preservation of substances which are normally subject to microbial spoilage. The objects of the invention include the provision of novel processes and compositions for accomplishing such preservation. Further objects of the invention will be evident from the following description wherein parts and percentages are by weight unless otherwise specified. The abbreviation ppm used herein refers to parts per million on a weight/volume (w/v) basis. Temperatures. are given in degrees Centigrade. The symbol (1) is used to designate the phenyl radical.

In accordance with the invention, it has been found that certain agents exhibit unexpected antimicrobial activity and are useful for preserving all kinds of materials which are normally subject to microbial spoilage. The agents in question are certain dihydrocinnamyl phenols, and their antimicrobial activity is unusual and unexpected because it is not shared by closely-related phenol derivatives.

Generically, the agents in accordance with the invention have the structure where at least one but not more than 3 of-the Rs are hydroxy, and the remainder of the Rs are each independently selected from the group consisting of lower alky. lower alkoxy, and hydrogen.

The compounds of the invention are especially useful because they are active against many microorganism, including those in the categories of bacteria, yeasts, and molds. In other words, the compounds are not just active against one or a few organisms; rather, they display broad-spectrum antimicrobial activity.

Examples of particular compounds within the scope of the invention are given below by way of illustration and not limitation:

Oil

Dihydro--tinnamyl-phenol (CH a IJihydro-Z-cinnamyl-phenol Dihydro-4-cinnamy1- 2-methylphenol Dihy dro- 2- cinnamyl- 4-ethy1phenol 4O Ilzthydro-Z-cinnamyl i-n-propylphenol Dihydro-Z-cinnamyll-i sop ropylphenol CH2-CH2-CH3 CI'KCHa) 2 (CH ca Dihydro-4-cinnamyl- 3 2 3-methyl- 6-1sopropylphenol 3 Oll L l (cup -as ocn a @flMi Dihydro- 2-c innamylr-me thoxy-phenol Dihydro- 4-cinnamy 1- 2-me thoxyphenol Dihydro-Z-cinnamyl- S-me thoxy-quinol (CH a Cll 0 The invention encompasses not only the use of any of the above-described agents individually, but also mixtures thereof.

In preserving substances in accordance with the invention, any of the aforesaid agents or mixtures thereof are incorporated with the substance, using an amount of the agent to inhibit microbial growth. Additional conventional treatments such as dehydration, canning, refrigeration, or freezing may be applied'to the substance containing the added agent. The incorporation of the agent with the substance may involve a mixing of the substance and the agentthis is especiallysuitable where the substance is in liquid or particulate form. Where the substance is in the form of pieces of large dimensions the agent may be incorporated therewith by coating it on the surface of the pieces. For such purpose the agent is preferably dispersed in a carriera liquid such as water, alcohol, water-alcohol blends, oils, or a finely-divided solid such as salt, starch, talc, or the like.

The invention is of wide versatility and can be applied for the preservation of all kinds of substances which are normally subject to microbial spoilage. Typical examples of such substances are listed below by way of example. Foodstuffs such as fruits, vegetables, juices, milk, eggs, meat, fish, grains, cereal products, cheese, etc. Animal glues and mucilages; dextrins; starch pastes and solutions; cosmetic, medicinal, and dental preparations; vitamin preparations; pastes, solutions, or other preparations of natural gums such as tragacanth, Arabic, acacia, karaya, locust bean, agaragar, pectin, algin, etc.; fermentation broths, mashcs, and residues from fermentation processes; whey; wines and Vinegars; animal feeds and ingredients of animal feeds such as fish meals, blood meals, feather meal, meat scraps, bone meal, tankage, grains, and oil-seed meals; proteins and protein hydrolysates, textile printing pastes; paints containing proteins or other spoilable dispersing agents; solutions of bark extracts or other tanning agents; molasses; by-products or wastes that contain potentially valuablecarbohydrate, p'roteinous or fat ingredients such as stick liquor, corn steep'liquor,

fruit cannery wastes, citrus peels, cull fruit and vegetables, tops of root vegetables, distillers slops. -pulp liquors, wash water from textile de-sizing operations, waste liquors from wool scouring plants, dairy and slaughter house wastes and liquors, etc.

The compounds of the invention display activities which are equal or even superior to those of widelyused antimicrobial agents. This is illustrated by the following: In general, the compounds of the invention are superior to such agents as phenol, resorcinol, o-phenylphenol, and the alkali metal sorbates and benzoates. For instance, the compounds of the invention, at concentrations of l2 to 25 ppm, were found to inhibit the growth of four bacteria (Bacillus cereux, Sarcinu lutea, Staphylococcus aureus, and Streptococcus lactis), whereas to achieve the same effect it required I00 to 200 ppm of o-phcnyl'phenol, more than 800 ppm of potassium sorbate, and more than 1,000 ppm of sodium benzoate.

Alky 4-hydroxybenzoates are well known to exhibit potent microbial activity. In general, the compounds of the invention display a superior activity as compared to these benzoates wherein the alkyl group contains less than seven carbon atoms. For instance, whereas the compounds of the invention at a concentration of 12 to 25 ppm will inhibit the growth of the four bacterial noted above. it requires concentrations of 100 to 400 ppm for the same result to be achieved with the C -C alkyl 4-hydroxybenzoates. Moreover, it may be noted that the compounds of the invention have and advantage in that their water-solubility is higher than that of the higher alkyl (e.g., heptyl) 4-hydroxybenzoates, and that the compounds of the invention do not exhibit the strong and unpleasant odor which characterizes the said benzoates. Also, with respect to some microorganisms, the compounds of the invention inhibit growth when applied at levels less than required with the higher alky 4-hydroxybenzoates. This is the case, for example, with bacterial such as Acaligenes faecelis and E. coli, yeasts such as Pic-Ilia choa'ati, Hansenula anomulu, and .Sut'c/uirmuycm cervvisiae, and molds including Aspvrigillus flui'us, A. uiger, Pc'nicillium chrysogcnum. Rhizupus semi, Bolrytis ('inerea, Byssochlamys fuli'u. and Allurnuriu .s'p.

Of the various compounds included within the scope of the invention. the dihydrocinnamyl-substituted monophenols display particularly high antibiotic activity against a large variety of different microorganism, and therefore are preferred with respect to the dihydrocinnamyl derivatives of dior triphenols.

As evident from the explanation immediately following Formula labove. the invention includes compounds wherein there may be nuclear lower alkyl or lower alkoxy substituents in addition to the hydroxy groups. In general. the compounds are preferred wherein such additional substituents are absent or, if present, are in small number, e.g., a total of one to two lower alkyl and/or lower alkoxy substituents. Taking the foregoing facts into consideration, we prefer to employ the compound of the sub-generic category represented by the formula wherein R is lower alkyl,

R" is lower alkoxy,

n is an integer from 0 to 2.

m is an integer from 0 to 2, and

the sum of n and m is not more than 2.

The compounds of the invention may be synthesized by known procedures. A typical plan is to first prepare a cinnamyl-substituted phenol, and then to hydrogenate this intermediate to form the dihydrocinnamylsubstituted phenol.

The intermediate may be prepared by the procedures of Hurd et al., Jour. Am. Chem. Soc., Vol. 59, pp. 107-109; Barnes, et al., Tetrahedron, Vol, 21, pp. 2,707-2715; Jurd, Experientia, Vol, 24, pp. 858-860; .lurd, Tetrahedron, Vol. 25, pp. 1,407-1416; and Jurd, Tetrahedron Letters No. 33, pp. 2,63-2866. Typically, these syntheses involve condensing a selected phenol (phenol itself, a cresol, resorcinol, guaiacol, hydroquinone monomethyl ether, etc.) with cinnamyl bromide or einnamyl alcohol.

In a next step, the cinnamyl-phenol intermediate is subjected to standard procedures to hydrogenate the ethylenic linkage whereby to produce a dihydrocinnamyl-phenol. Generally, this involves treating the cinnamylphenol with hydrogen gas in the presence of a suitable catalyst, such as platinum oxide, palladium or charcoal, and the like. Typically, 10-20 grams of a cinnamylphenol is dissolved in 50-100 milliliters ofa nonhydrogenatable organic solvent, such as acetic acid, tetrahydrofuran, and the like, and 0.1 to 1.0 gram of the catalyst is added. Then, hydrogen gas is applied at approximately 30-50 pounds per square inch. The dihydro derivative can be purified by common techniques, for example, distillation or crystallization.

In the copending application of Leonard Jurd et al., Ser. No. 74,485, filed Sept. 22, 1970, issued July 10, 1973, as US. Pat. No. 3,795,222, there are described certain cinnamyl phenols which are useful as antimicrobial agents. The said cinnamyl phenols have the structure R CHZ-CH cuwherein at least one but one but not more than three of the Rs are hydroxy radicals, and the remainder of the Rs are each independently selected from the group consisting of lower alkyl, lower alkoxy, and hydrogen. It should be noted that the compounds of Ser. No. 74,485 contain the unsaturated side-chain CH CH CH, whereas in the compounds of the present invention the corresponding substituent is the saturated group -CH CH CH As such, the instant agents represent a class of compounds totally separate and distinct from those of Ser. No.-74,485. Moreover, the compounds of the presentinvention provide an advantage in that they are more stable. Since they do not contain ethylenic unsaturation, they are not subject to oxidization when contacted with oxygen, air, or other oxidizing media.

EXAMPLE The invention is further demonstrated by the following illustrative example.

A series of compounds was assayed for effectiveness against bacteria, yeasts, and molds, using the following test procedure.

All compounds were initially tested at a eoncentration of 200 or 500 ppm. in some cases. assays also were conducted with varying amounts of the compounds to determine the minimum concentration thereof required to inhibit growth.

Plates were prepared by adding a measured amount of the candidate compound in acetone solution to 10 ml. of sterilized medium, mixing thoroughly, pouring into 60 X 15 mm. plastic petri dishes, and allowing the gel to set and age overnight. The plates were then inoculated with the test organisms. In the case of bacteria and yeasts, the inoculation was done by the Lederberg and Lederberg (J. Bacteriol., 63, 399, 1952) replica plating technique, applying nine bacterial or seven yeasts on each plate. In the case of molds, drops of homogenized culture were placed on the surface of the plates, applying three or four molds per plate. The media used were: plate count agar (Difco), pH 7.0, for

cinnamyl, and Pl-l" for phenol. Thus, for example, dihydro-4-cinnamyl-phenol is designated as DH-4-CA- PH; dihydro-2-cinnamyl-4-isopropylphenol is designated as DH-2-CA-4-isopr0pyl-PH.

TABLE I Minimal inhibitory Concentrations (in ppm) of Dihydrocinnamyl Phenols on Growth of Gram-positive Bacteria The symbol indicates complete inhibition of growth at a concentration of 500 ppm. The

compound in question was not tested at lower concentration.

bacteria; and potato dextrose agar (Difco), pH 5.6, for yeasts and molds. Inoculated control plates were also prepared with media containing the largest amount of acetone (0.8 ml.) used in the preparation of the test plates. We have determined that acetone has a minimal influence on the growth of organisms in this procedure. The plates were incubated at 28 for ldays and eval- TABLE ll Minimal Inhibitory Concentration (in ppm.)

of Dihydro-4-cinnamyl Phenol on Growth of Gram-negative Bacteria Minimal inhibitory conc.

30 Bacteria of DH-4-CA-PH, ppm.

nated by comparison with the controls. Alcaligenesfaet'ulis 8-170 200 '7 The results obtained are summarized in the following m 588 tables. In these tables the compounds are designated in salmonella rypliimurium Tm l 200 abbreviated manner using DH for dihydro, CA" for 56mm 200 TABLE III Minimal Inhibitory Concentrations (in ppm.) of

Various Dihydroeinnamyl Phenols on Growth of Yeasts Pic/tin Saccharmnyces c'lmdafi cererisiae Torula Z \'gosuzchar0m \'(3 Candida var. var. uliIis jupunicus tropicalis fermenmns Hunsenulu ellipsaideus NRRL Compound G124 G147 G238 anomala Y-44 Y-660 DH-4-CA-PH 25 25 5O 25 25 DH-Z-CA-PH l2 I2 50 25 12 25 DH-2-CA-4-methyl-PH l2 l2 25 12 DH-4-CA-2-methyl-PH l2 l2 200* I00 12 200 DH-2-CA-4-ethyl-PH 12-25 12-25 50 lOO DH-2-CA-4-methoxy-PH 25 25 50 25 200 qhh symbol indicates no growth inhibition at cone. of 500 ppm. The term 200" indicates growth not inhibited with 200 ppm. (highest concentration tested in t is case).

TABLE IV Minimal Concentrations (in ppm.) of Various Dihydrocinnamyl Phenols on Growth of Molds Com pound Aspergillus Axpergillus Penicillin!" Rhizapus Botrylis B yssochlum ys Allt'rnuria flurus niger cltrysogenium .renli tinert'a Iva .s'p. NRRL NRRL NRRL NRRL 3145 A-7705 52 2868 3492 3493 DH-4-CA-PH 200 50-l00 25 50 12-25 50 25 DH-Z-CA-PH 5O 50 25 25 12 25 25 DH-2-CA-4-methyl-PH 200* 200 200 lOO 200 200 lOO DH-4-CA-2-methyl-PH 200 200 25 50 25 25-50 25-50 DH-2-CA-4-ethyl-PH 200 -200 200 200 DH-2-CA-4-isopropyl-PH 200 200 200 100 DH-2-CA-4-methoxy-PH 200 100 200 200 200 The symbol indicates no growth inhibitation at concentration of 500 ppm. The term 20U" indicates growth not inhibited at 200 ppm. (highest concentration tested in this case),

l. A process for inhibiting the growth of microorganisms selected from the group consisting of bacteria,

yeast, and molds in a substance normally subject to spoilage by said microorganisms, which comprises applying to the substance an effective microbial-growthinhibiting amount of a compound of the structure: wherein l to 3 of the Rs are hydroxy, and the remainder of the Rs are each independently selected from the group consisting of lower alkoxy and hydrogen wherein at least one of the Rs is lower alkoxy.

2. The process of claim 1 wherein the compound is dihydro-Z-cinnamyl-4-methoxyphenol. 

1. A PROCESS FOR INHIBITING THE GROWTH OF MICROORGANISMS SELECTED FROM THE GROUP CONSISTING OF BACTERIA, YEAST, AND MOLDS IN A SUBSTANCE NORMALLY SUBJECT TO SPOILAGE BY SAID MICROORGANISMS, WHICH COMPRISES APPLYING TO THE SUBSTANCE AN EFFECTIVE MICROBIAL-GROWTH-INHIBITION AMOUNT OF A COMPOUND OF THE STRUCTURE:
 2. The process of claim 1 wherein the compound is dihydro-2-cinnamyl-4-methoxyphenol. 